Project Summary/Abstract The repair of cartilage injuries still remains a major challenge. Despite the advancement of surgical techniques, including microfracture surgery and cell-based approaches, all of these approaches do not form hyaline cartilage but rather `hyaline-like' cartilage or fibrocartilage. Since the function of articular cartilage is linked to its structure, all current surgical interventions to repair cartilage defects result in some impairment and the development of post-traumatic osteoarthritis (PTOA). Therefore the development of novel therapeutic strategies for cartilage repair is highly warranted. Our preliminary data suggest that hyaluronan (HA), a major component of the cartilage extracellular matrix (ECM) together with a RHAMM-mimetic, HA-binding peptide (P15-1) play a major role in the settlement of mesenchymal stem cells (MSCs) at the cartilage repair site and their chondrogenic differentiation. This peptide inhibits the interaction of HA with one of its receptors, receptor for hyaluronan-mediated motility (RHAMM). RHAMM associates with another HA receptor CD44. P15-1 prevents the interaction between HA and RHAMM and allows the clustering of CD44 by HA. Our collaborators who discovered P15-1 have shown that HA/RHAMM/CD44 interactions stimulate stem cell migration and proliferation but also fibrotic processes, including fibrotic tissue healing, whereas interfering with HA/RHAMM interactions with P15-1 promoted a form of scarless skin wound healing. Our preliminary findings showing a markedly improved cartilage repair of full thickness cartilage defect in rabbits after microfracture surgery and three intra-articular injections of P15-1 together with high molecular weight (HMW)HA compared to intra- articular injections of HMWHA alone, and a markedly improved attachment of bone marrow-derived mesenchymal stem cells (BMSCs) to tissue culture plates coated with HMWHA and P15-1 compared to plates coated with HMWHA only support our hypothesis that interfering with HA/RHAMM interactions in MSCs will enhance settlement of these cells at the repair site and their chondrogenic differentiation leading to enhanced cartilage repair. To test this hypothesis we propose two Aims. In Aim 1 we will perform in vitro experiments using P15-1, antibodies that block HA/RHAMM and/or HA/CD44 interactions, RHAMM knockout (- /-) BMSCs, and CD44-/- BMSCs to determine how the interactions of HA with RHAMM and CD44 affect BMSC proliferation, migration, attachment to HA and chondrogenic differentiation. In Aim 2 we will confirm our in vitro results and determine whether interfering with HA/RHAMM interactions via intra-articular injections of P15-1 together with HMWHA and BMSCs will lead to enhanced settlement of BMSCs at the repair site and improved formation of hyaline cartilage. Finally, we will determine whether injections of BMSCs together with P15-1 and HMWHA are more effective in cartilage repair than microfracture surgery and intra-articular injections of P15-1 and HMWHA. The successful completion of this grant is expected to establish a novel therapeutic strategy to improve cartilage repair.